Lecture 9 C. elegans cell biology C. elegans genetics C. elegans genome.

Lecture 9

• C. elegans cell biology

• C. elegans genetics

• C. elegans genome

Theoretically perfect model organism

• Well characterized life cycle-all stages easily accessible.

• Well characterized genetic system. • Well characterized genome-basically

sequenced and annotated.• The ability to reintroduce DNA into the

organism-transgenesis.• Closely related to humans-funding purposes.

Caenorabditis elegans• Life cycle: short 3 1/2 days, transparent

organism, complete cell lineage known.

• Genetic system: both classical and RNAi

• Genome: first metazoan sequenced 1998

• Transgenesis: injection of DNA

• Related to humans?

Caenorabditis elegans

Hermaphrodite

Horvitz and Sternberg Nature 351, 535

Hermaphrodite and male

Wood 1998 The Nematode C. elegans

Cross sectiontube within a tube

Wood 1998 The Nematode C. elegans

Wood 1998 The Nematode C. elegans

Life cycle

Wood 1998 The Nematode C. elegans

Life cycle

Hermaphrodite558 nucleiMales560 nuclei

Wood 1998 The Nematode C. elegans

Hermaphrodite and male gonadogenesis

Wood 1998 The Nematode C. elegans

Life cycleHermaphrodite959 somatic nucleiMale1,031 somatic nuclei

Wood 1998 The Nematode C. elegans

Worm’s brain

White et al. Phil. Trans. Royal Soc. London 314, 1-340

All neuronal connections known

White et al. Phil. Trans. Royal Soc. London 314, 1-340

Hermaphrodite and male

Wood 1998 The Nematode C. elegans

Fertilization and the first divisions

Kalthoff Analysis of Biological Development

Complete cell lineage

Slack and Ruvkun Annu. Rev, Genet. 31, 611

Cell lineage

• Early divisions

• Lineage structure and nomenclature

• Cell death

• Repeated lineages

First four divisions and major blast cells

Wood 1998 The Nematode C. elegans

First four divisions and major blast cells

Complete cell lineage

Slack and Ruvkun Annu. Rev, Genet. 31, 611

Wormbase

Temporal and spatial information

AB

time

AB.a AB.p

M.vlpaa

Key blast cells are given upper case letters

The progeny are named by adding lower caseletters indicating the division axis:a-anteriorp-posteriord-dorsalv-ventrall-leftr-right

M great great great grandmothM.v great great grandmotherM.vl great grandmotherM.vlp grandmother M.vlpa motherM.vlpaa daughter

Following the lineage

Cell death

AB.alaaaala

alaralal

l r

DEADNeuron in ring ganglion

Kalthoff Analysis of Biological Development

Repeated lineages

Wormbase

Repeated lineages

Wormbase

How is cell fate determined?

English vs American view

Complete cell lineage

Slack and Ruvkun Annu. Rev, Genet. 31, 611

Fertilization and the first divisions

Kalthoff Analysis of Biological Development

How is cell fate determined?

English vs American view

Experimental approach: laser cell ablation

Nonautonomous determination

• Induction

• Equivalence groups

Induction

1

2

A cell or group of cells removedfrom a second cell

that directs the developmentalfate of a second cell or group ofcells.

Example of induction

Anchor cell-gonad

signals

Epidermis

Vulva

Repeated lineages

Wormbase

Equivalence groups: Group of cells that have equivalent pluripotent cell fates.

Z1.ppp Z1.pppZ4.aaa Z4.aaa

Individual A Individual B

Anchor cell/ Ventral uterine cell equivalence group

AC ACVU VU

Z1.ppp Z1.pppZ4.aaa Z4.aaa

Experiment A Experiment B

Anchor cell/ Ventral uterine cell equivalence group

AC AC

Cell ablation experiment

Z1.ppp Z1.pppZ4.aaa Z4.aaa

Experiment A Experiment B

Anchor cell/ Ventral uterine cell equivalence group

AC AC

Cell ablation experiment

The remaining cell always becomes an AC.The AC fate is the 1° (primary) cell fate.

Vulva equivalence group

Wormbase

P3.p P8.p

X X XY YZ

Vulva equivalence group

P3.p P8.p

X X XY YZ

Vulva equivalence group

X XY YZ

X XY YZ

Z

Vulva equivalence group

Z is the 1° cell fate

Y is the 2° cell fate

X is the 3° cell fate

C. elegans genetics

1. Self-fertilization

2. Systematic approach with RNAi

Self-fertilization and homozygousity

m/+ F0

m/m m/+ +/+ F1

m/m m/+ +/+ F2

Self the population

Mutagenesis and screens

P0young hermaphrodite

EMS

+/+ +/+ +/+ +/+ +/+ +/m +/+ +/+ ….. F1

self self

F2All wild-type

Males

X X hermaphroditeX O male

At a frequency of 1/1000, males arise due to nondisjunction of the X chromosome.

Complementation analysis

males m1/m1 X hermaphordites m2/m2

Look at males only?

Complementation analysis

males m1/m1 X hermaphordites m2/m2

1. All males have mutant phenotype

2. All males are wild-type

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

a- m+

a+ m-

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

Somea-

a- m+

a+ m-

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

Somea-

a- m+

a+ m-

a- m+

a- m+

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

Somea-

a- m+

a+ m-

a- m+

a- m+

Rarem-

Non complementation screen

male a+ m-/a+ m- X hermaphrodite a- m+/a- m+

EMS

MostWild-type

Somea-

a- m+

a+ m-

a- m+

a- m+

Rarem-

a- m-new

a+ m-

Transgenesis

YFG

rollD

Look for rolling progeny F1

Horvitz and Sternberg Nature 351, 535

Transgenesis

YFG

rollD

Look for rolling progeny F1

Look for rolling progeny in F2

Horvitz and Sternberg Nature 351, 535

Transgenesis

Nucleus of F2 rolling progeny

YFG rollDYFGYFG rollD rollD rollD

Large concatenated arrays that are stablely maintained.

NCBI

RNAi inhibition of gene expression

1. RNAi discovered in C. elegans and plants.

2. Double stranded RNA results in the degradation of homologous mRNA.

3. Double stranded RNA can be fed to worms in the E. coli they eat.

4. Allows for the systematic inhibition of all 20,000 genes of C. elegans.

Systematic RNAi screens in C. elegans

Tuschl Nature 421, 220